Comparison of PetrifilmTM AC and pour plate techniques used for the heterotrophic aerobic bacterial count in water

Abstract Heterotrophic bacteria are commonly found in water samples. While these Heterotrophic Bacterial/Plate Counts (HPC) do not necessarily indicate a health hazard, high counts provide a good indication of the efficiency of water disinfection and integrity of distribution systems. The aim of this study was to compare the PetrifimTM AC method to the pour plate technique for the testing of HPC in water samples. Artificially contaminated (192 samples) and natural water samples (25) were processed using two methods. Both methods accurately detected high, medium and low counts of HPC, producing average Z scores between −2 and +2. Paired-wise student t-test and correlation coefficient showed nonsignificant differences between the results of two methods. Acceptable repeatability and reproducibility was obtained using both the methods. Uncertainty of measurement for PetrifilmTM AC and pour plate method was found to be 2.9% and 5.4%, respectively. PetrifilmTM AC proved to be robust at 33°C and 37°C. In conclusion, PetrifimTM AC, which is easy to process, read, and less time consuming, proved to be comparable to the conventional pour plate method in establishing HPC in water. In addition, PetrifimTM AC requires less space for the processing and incubation, generate small volume of waste for disposal, and requires no equipment, except for the incubator.


Introduction
According to the World Health Organization (WHO) drinkingwater should be suitable for human consumption, washing/sho w ering, and the pr epar ation of food.Exposur e to water and its constituents can occur through ingestion, contact, and inhalation.Ther efor e, drinking water should be micr obiologicall y safe (WHO 2023 ).In 2022, according to WHO, 6 billion people used safel y mana ged uncontaminated drinking water and 2.2 billion people used untreated water.While the untreated water is known to transmit pathogens; with a mismanagement of treated water and suppl y c hain br oken, tr eated water can also become a source of infection (WHO 2023 , UN-Water 2021 ).For example, a systematic r e vie w containing studies fr om high-income countries mainl y from north America, sho w ed that the vast majority of residents ( ≥90%) r eportedl y hav e high access to safel y mana ged drinking w ater, and y et the bur den of w ater-r elated gastr ointestinal illness risks was ∼2720 annual cases per 100 000 population (Lee et al. 2023 ).
Micr oor ganisms can gr o w in w ater and on surfaces in contact with water, such as distribution pipes, domestic plumbing, and v ending mac hines.Commonl y found micr oor ganisms ar e heter otr ophs, whic h r equir e or ganic carbon for gr owth.These include bacteria, yeasts, and mould.A wide range of micr oor ganisms, whic h r equir e v aried nutrients , physiological conditions , and incubation time .T her efor e, at a giv en time and growth condition used, micr obial r ecov ery will include a fr action of natur al micr obiota of water, as well as contaminants (Allen et al. 2004 ).
With regards to the total heter otr ophic plate counts (HPC) in treated potable water, countries have different directives (Saxena et al. 2015 ), and the acceptable counts varies from 10 to 1000 cfu/ml of water.In gener al, HPC hav e no health burden and the test cannot stand alone .Additional tests , such as total coliform or the counts of faecal coliforms and Esc heric hia coli counts, ar e r equir ed to determine the health hazard.Ne v ertheless, counts of heter otr ophic bacteria provides a good indication of efficiency of water disinfection and the integrity of distribution systems.Constant surveillance can show early detection of possible presence of external contamination of water (WHO , EP A 2024 , Canadian drinking water, EN dir ectiv e 2020 ).A v ery high number of heter otr ophic bacteria, ho w e v er, does indicate possible water contamination and the possible presence of health hazards.In South Africa, drinking water guidelines and national standards allows Heter otr ophic Bacterial/Plate Count (HPC) of ≤1000 cfu/ml in drinking water (SANS 241-1 2015 ).Counts higher than that are described to have increased risk of infectious disease transmission, particularl y r espir atory infections including Legionella, and opportunistic infections in imm unocompr omised individuals, and nosocomial infections including wound infections, urinary tract infections, and postoper ativ e infections.In addition, high counts of HPC can lead to biofilm formation in pipes leading to further establishment of micr oor ganisms, whic h can also cause foul taste and aestheticall y r educed quality of water (Allen et al. 2004, WHO 2022 ).Many methods are described for the enumeration of HPC, such as membr ane filtr ation, pour plate, spr ead plate, and fluor escent substrate technique, each with advantages and disadvantages (Allen et al. 2004 ).The pour plate method is most commonly used (Shakoor et al. 2018, Amin et al. 2019 ).Ready-to-use dehydrated media have been developed and commercialized as Petrifilm TM AC.It is widely used for total bacterial counts in food and milk samples.Petrifilm TM AC, which is used for aerobic bacterial counts, has not been tested and validated for the water samples .T he aim of this study was to compare the conventional pour plate technique to Petrifilm TM AC and validate the Petrifilm TM AC to test water samples for the total bacterial counts.

Water samples and spiking with bacteria
Chlorinated tap water was collected from a drinking water tap into 1 l glass bottles.Borehole water (1 l) was collected from the P arktown, Johannesbur g r egion.Bottled water was obtained from a supermarket.Tap water and borehole water samples were autoclaved.Sodium thiosulfate (1%), which is generally used to neutralize the residual chlorine in the samples, was added to one set of tap water.This was to establish if it had any effect on the final HPC results.
Cultures of E. coli ATCC 25922, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 21922, and Candida albicans ATCC 90028 were obtained from the Infection Contr ol labor atory, National Health Labor atory Services (NHLS), South Africa.For the use of these stock cultures, an ethical waiver was obtained from the Human Research ethic Committee, University of The Witwatersrand (W-CBP-230711-01).Raw material for the culture media and r ea gents wer e purc hased fr om Merc k, South Africa.Cultures were grown on blood agar plates at 37 • C for 24-48 h, and purity was established.One isolated colony from each of the culture plates was emulsified into 10 ml sterile distilled water to obtain McFarland standard of 0.5 containing ∼10 8 cfu/ml.Serial 10-fold dilutions (up to 10 −6 ) were prepared using 9 ml buffered peptone water.Dilutions 10 −3 , 10 −4 , and 10 −5 were selected for further testing.Original suspension of 0.5 was also serially diluted and each dilutions were plated onto TSA agar plates to obtain an accurate count total bacteria, which were considered as expected counts.
Water samples were inoculated with selected dilutions to obtain ∼1000, 100, and 10 cfu/ml of water samples r epr esenting high, medium, and low quantities of test counts.Uninoculated samples were also processed as negative controls.Sterility of all the media, including P etrifilm TM AC, w as established.

3M TM Petrifilms aerobic plate count method (AC)
Tests were performed as per manufacturer's instructions (3M, South Africa).Two plates per sample were used.Briefly, 1 ml of sample was pipetted per pendicularl y into the centre of the bottom film containing dehydrated media and spread using a spreader as provided with the P etrifilms TM AC.Plates w ere k e pt on the bench for 15 min and allo w ed to solidify and set.Plates were incubated at 35 ± 2 • C for 48 h in a closed container with a moistur e pac k to pr e v ent dehydr ation.Humidity of these containers was not measured in accordance with manufacturer's instructions.Red colonies were counted regardless of size and intensity.Preferred counting range was 10-300 cfu/ml, howe v er, colonies mor e than the upper range were counted as estimated counts.Number of colonies in one square were counted and multiplied by 20 to obtain the total number per plate, hence 1 ml of sample .T he a v er a ge of the counts of the two plates was taken as a final count.

Pour plate method
Pour plate method is one of the oldest techniques used for the total bacterial counts.Briefly, 1 ml of sample was placed in the centre of a sterile Petri dish, molten cooled (45 • C) plate count agar (Mer ck, South Africa) w as pour ed ov er it and mixed gentl y.The media w as allo w ed to solidify and set.Plates w er e inv erted and incubated at 37 • C for 48 h (Jackson et al. 2000 ).Clear, white, and opaque colonies on the surface of the agar as well as into the agar at v arious le v els, wer e counted.Two plates per sample were used.T he a v er a ge of the counts of the tw o plates w as taken as a final count.

Validation procedure
The v alidation pr ocedur e described in TR 28 document was used with minor modifications (TR 28-01 2014 ).This document defines the technical requirements for quality control, quality assurance, and the validation of methods in microbiological testing laboratories.Accurac y w as established b y calculating Z scores using the complete data (16 results + inoculum).An acceptable range was set at −2 to + 2. Repeatability was calculated using se v en r esults obtained by one analyst on the same day.Reproducibility was cal-

Na tur al samples
Natural samples of potable water ( 25) received by the Public Health Labor atory, NHLS, Johannesbur g for r outine testing wer e processed in parallel using both the methods.Results were analysed using correlation coefficient.

Participation in an external quality assurance
To further establish the reliability of the Petrifilm TM AC, our laboratory participated in an external quality assur ance, wher e samples were provided by Axio QWAS distribution, LGC Profeciency testing, 1 Chamberhall Business Park, Chamberhall Green Bury, UK.In total, 21 distributions were processed using the Petrifilm TM AC method between November 2017 and June 2023.The av er a ge number of laboratories participated per distribution is 204 and the r esults wer e anal ysed b y the distributor.Results w er e r eceiv ed in the form of Z scores, with an acceptable range between −2 and + 2.

Accuracy and the comparison of both methods
Results of spiking inoculum (expected r esults) wer e compar ed to the results obtained by all four analysts .T his was performed for all the matrices, two methods, and three concentrations.All the counts were converted into log 10 and analysed.Mean ± SD counts for Petrifilm TM AC for the high, medium, and low concentr ations wer e 3.08 ± 0.13, 2.44 ± 0.12, and 1.68 ± 0.13, respectiv el y.Wher eas, for the pour plate method, they were 3.14 ± 0.13, 2.45 ± 0.12, and 1.58 ± 0.16, r espectiv el y.The Z scor es of eac h test r esults wer e found to be between −2 and + 2 (Table 1 ).This suggested that both the methods produced accurate results for all the matrices at high, medium, and low concentration of bacteria.When the results obtained with P etrifilm TM AC w ere analysed with the results of pour plate technique, it was shown that the results are comparable with mean Z-score between −2 and + 2 (Table 2 ).The correlation coefficient (0.95) was acceptable ( Figure S1 and Table S1 ) and the paired t -test sho w ed that there was no difference in the counts obtained from the two methods ( P > .05).

Repeatability and reproducibility
Re peatability and re producibility are important as it indicates that the methods ar e r obust.If the confounding factors and competencies of oper ators ar e consider ed, it pr oduces accur ate r esults.For the r epeatability, r esults obtained by one analyst (seven readings) for all four matrices, both the methods, and all three concentrations (high, medium, and low) were analysed for RSD.The results were found to be in an accepted range of ≤0.1 RSD as per Table 3 .For the r epr oducibility, r esults obtained by three ana-l yst on thr ee differ ent days wer e compar ed and anal ysed for r elative standard deviation (RSD RC ).Acceptable mean RSD RC of ≤0.1 were obtained for both the methods (Table 4 ).

Uncertainty of measurement and robustness
Uncertainty of measurement was calculated using results of reproducibility (RSD RC ) for both the methods including all the matrices .T hese results may vary from laboratory to laboratory.In our laboratory, for Petrifilm TM AC, with counts of 100 cfu/ml, the uncertainty was found to be between 87 and 114 cfu/ml of water (2.9%).For pour plate, with counts of 100 cfu/ml, the uncertainty was found to be between 78 and 128 cfu/ml (5.4%).Robustness was determined only for the Petrifilm TM AC method because the aim of this study was to validate this method.Petrifilm TM AC tests were performed in two duplicate sets.One set was incubated at 33 • C and the other at 37 • C. The rationale was determine the effect on final results should the incubator temper atur es fluctuate b y 2 • C either w a y.T he results sho w ed that there was no difference in the results obtained at 33 • C and 37 • C in Petrifilm TM AC (Table 5 ).

Effect of sodium thiosulfate
When the results of water samples, with and without sodium thiosulfate, wer e compar ed, no significant differ ence in the HPC was found; with P -values of > .05(Table 6 t)

Na tur al samples
Out of 25 samples, 15 samples colony counts were impossible to count using both the methods (too numerous to count).A total of 10 samples pr ovided measur able counts .T he mean count and the range for P etrifilm TM AC w as 167 cfu/ml and 1-710 cfu/ml, r espectiv el y.Wher eas, for the pour plate method, the mean and r ange wer e 176 cfu/ml and 5-736 cfu/ml, r espectiv el y.For these 10 results, the correlation coefficient was 0.99 ( Table S2 ).

Participation in an external quality assurance
All 21 rounds of distribution received acceptable Z scores between −1 and + 1 using Petrifilm TM AC ( Figure S2 ), further establishing accuracy of the Petrifilm TM AC method.

Discussion
Aerobic plate count, which is a synonym for heterotrophic plate count or total bacterial counts, has a long history of use in water micr obiology.HPC measur ements indicates the effectiv eness of water treatment processes, post-treatment contamination, and r egr owth of organisms .T he pour plate technique is one of the oldest methods de v eloped for the determination of HPC in water.Media containing basic nutrient, such as tryptone and yeast extr act is used, whic h supports the growth of wide variety of bacteria that r equir e or ganic carbon for gr owth.It is an effectiv e, v alidated, well-r ecognized, and widel y used method.Ho w e v er, it can be time consuming especially in very busy laboratories that process high volumes of water samples.3M TM has de v eloped dehydr ated a gar plates containing a water-soluble gelling agent, nutrients, and indicator that facilitates enumeration of HPC.A built-in grid facilitates counting of colonies, to provide fast, precise, and consistent results.It has been extensively used for testing food samples, ho w e v er, it has not been studied for the testing of water samples.
Results in this study sho w ed that Petrifilm TM AC method is comparable to the pour plate method, with the Z scor e ar ound zer o when the results of both the methods were analysed together for the high, medium, and low counts .T he correlation coefficient was also acceptable at 0.95.In addition, international proficiency testing results were also comparable to ∼200 laboratories that used man y differ ent methods.In contr ast, when Sc hr aft and Watterworth ( 2005 ) compared the Petrifilm TM AC method to the membr ane filtr ation to test water, they found a significant difference in the counts, with the counts on Petrifilm TM AC being 0.5 log lo w er than membrane filtration technique.In their study, only natural samples wer e pr ocessed, wher eas in our study, sterile samples were inoculated with a mixture of organisms to cover a range of counts, as well as natural samples.
Although Petrifilm TM AC is validated for the food and milk testing and well-studied (McAllister et al. 1988, Curiale et al. 1990 ), it has not been studied for the water samples .T he reason could be, although HPC has significance in the water samples, there are no legislated acceptable counts available in many countries because it is not a standalone test (Bartram et al. 2003, Allen et al. 2004, Saxena et al. 2015 ).For potable waters, coliform and E. coli counts are required as additional tests.Several epidemiological studies have failed to establish the correlation between the HPC in drinking water and gastrointestinal infections (Calderon and Mood 1988, Payment et al. 1991, Hellard et al. 2001 ).This suggests that HPC is not a health-related indicator, but it is a process indicator (disinfection and reticulation).For the quality indicator of water service delivery, this test can easily be used.In South Africa, HPC r equir ement in the potable water is legislated with the acceptable counts of ≤1000 cfu/ml of drinking water, ther efor e this test is extensiv el y used locally (SANS 241-1 2015 ).Counts between 100 and 1000 cfu/ml ar e indicativ e of some contamination or inadequate treatment, but counts > 1000 cfu/ml has increased risk of infectious disease transmission (Department of Water Affairs and Forestry 1996 ), particularly respiratory infections and nosocomial infections.United States Environmental Protection Agenc y allo ws ≤500 cfu/ml of HPC in ground and drinking water (EPA).
Petrifilm TM AC is a simple method that r equir es v ery short pr ocessing time when compared to the pour plate technique (1-2 min vs. 30 min).Dehydr ated media is r eady to use and it r equir es less handling, thus r educing c hances of contamination compared to the pour plate method, where melting, cooling, and pouring of media is r equir ed.Petrifilm TM AC do not r equir e an y equipment.Since these plates are compact, laboratory space for processing and the incubator space r equir ed is also less than pour plate method.Thickness of Petrifilm TM AC is 0.1 cm, and therefore can be stacked in large quantities for incubation, whereas Petri dishes used for the pour plate have a thickness of 1.5 cm that r equir e mor e space during pr ocessing of samples and incubation.Waste disposal can also be expensive because it is calculated per weight of the waste.Petrifilm TM AC weighs 2.8 g with a sample whereas pour plate weighs 32.7 g.One of the additional disadvantages of the pour plate method is that the agar has to be used at pr ecisel y 45 • C, an y higher than that the temper atur e may kill some of the bacteria.Agar at lo w er temper atur es than 45 • C may start solidifying at room temperature, creating lumps and compr omising e v en distribution of colonies into the a gar, ther eby compr omising colon y counts.In Petrifilm TM AC, although the colon y counts are performed in 5-cm diameter of r ehydr ated media film compared to the 9-cm diameter surface of pour plate agar, counting colonies is easier due to the pink colour of the colonies.In the pour plate method, mostly white colonies are on the surface and submerged into the pale colour media, making it difficult to r ead the r esults.Although Petrifilm TM AC has a counting grid, in the case of higher counts, counting colonies can be a problem.Ther efor e, manufactur er's r ecommended accur ate count is Both the methods allow accurate counts up to 300 cfu/ml of sample , thereafter if possible , higher counts can be obtained by diluting the sample.Presence of bacteria that cause liquefaction of agar and those that have the ability to spread on the agar surface can create interference in counting colonies especially in the Petrifim TM AC (Blackburn et al. 1996 ).
Results also sho w ed that the repeatability and reproducibility of P etrifilm TM AC w as acceptable, meaning the r esults ar e not affected by the conditions and analysts.Robustness was studied using variation in only one condition (temperature) due to the resource constr aints.Ne v ertheless, Petrifilm TM AC pr ov ed to be acceptable in the presence of two incubation temperatures.Howe v er, Ellender et al. ( 1993 ) found higher HPC in food samples when P etrifilm TM AC w ere incubated at room temperature compared to the 35 • C incubation.Perhaps, the nutrients present in the food samples influenced the bacterial counts.Water samples gener all y are low in any form of nutrient, therefore the results were not affected.
In conclusion, Petrifilm TM AC pr ov ed to be compar able to the widel y used tr aditional pour plate method.With man y adv anta ges, Petrifilm TM AC pr ov ed to be an accur ate, r epeatable, r epr oducible, and robust method for establishing heterotrophic bacterial counts in water.

score ( n = 16) Matrix Concentr a tion Petrifilm TM Pour plate
Ta ble 1. Accurac y of the two methods for the aerobic plate count in water.

Table 2 .
Comparison and accuracy using combined results of both the tests for aerobic plate count test in water and linearity.

Table 3 .
Repeatability of Petrifilm TM and pour plate methods for the aerobic plate counts in water.

Table 4 .
Reproducibility of Petrifilm TM and pour plate methods for the aerobic plate count in water.

Table 5 .
Robustness of Petrifilm TM method for the aerobic plate count in water.

Table 6 .
Comparison of HPC in the water samples with and without sodium thiosulfate.

± SD cfu/ml ( n = 16) Method Concentr a tion Without Na 2 S 2 O 3 With Na 2 S 2 O 3 P -value
ml/plate .Abo ve these counts , colon y counts ar e possible but may not be accurate, and ther efor e r eported as an estimated counts.